Abstract Type 2 diabetes is associated with insulin resistance and disturbances in pancreatic cell function that result in inadequate glucose-stimulated insulin secretion (GSIS). However, the mechanisms that cause cell failure are largely unknown. Recent studies implicate protein misfolding in the ER as a potential cause for cell failure in diabetic humans. Upon accumulation of unfolded proteins in the lumen of the ER, PERK, IREI?, and ATF6? are activated to increase the capacity of the ER to meet the demand for increased protein folding and to increase the protein degradative machinery to eliminate misfolded proteins. In addition, protein synthesis is transiently attenuated through PERK-mediated phosphorylation of elF2?. Over the past cycle we demonstrated: 1) elF2? phosphorylation is required to limit protein synthesis and oxidative stress to maintain cell function; 2) the ER co-chaperone p58IPK is required to limit reactive oxygen species (ROS) and preserve cell function. Antioxidant treatment significantly restores cell function in p58IPK+/+ mice; and 3) IRE1?-mediated splicing of Xbp1 mRNA induces co-transiational translocation at the ER to promote proinsulin production and represses oxidative stress. The sum of our data lead us to propose that tight control of protein synthesis in the cell is required to ensure the ER protein folding demand does not exceed the capacity. This is especially important for the cell as it is exposed to periodic postprandial increases in protein synthesis. In our Specific Aims, we will test three hypotheses by answering the following questions: Aim 1: Translational attenuation through elF2? phosphorylation preserves cell function by limiting protein misfolding. We propose that excessive proinsulin synthesis causes proinsulin misfolding, ER Ca2+ release and uptake into mitochondria, and mitochondrial-generated ROS. ROS then feed forward to further disrupt protein folding in the ER. Any stimuli that pressure cells to exceed their capacity for proinsulin folding will succumb to this vicious cycle. To test this notion, we will answer: a. Does excessive proinsulin synthesis (such as elF2?AA) cause cell dedifferentiation and can; antioxidants protect cells under these conditions? We will sort GFP+ elF2?AA cells from mice (+/- BHA-supplemented diet) and characterize their gene expression and D N A replication/damage patterns. b. Can reduced protein synthesis protect cells in elF2ccAA mice? We will test whether decreased protein synthesis through haploinsufficiency in the ribosomal protein RPL24 gene can protect elF2?AA cells. c. How does elF2? phosphorylation change 5' open reading frame (ORF) usage in mRNAs? Ribosomal protection assays will be performed to elucidate how elF2? phosphorylation alters ORF usage in response to glucose stimulation in wildtype and elF2?AA cells. d. Can pharmaceutical interventions protect elF2?AA cells that produce excessive proinsulin? We will test chemical chaperones, GLP-1, cyclosporin A, rapamycin/carbamazepine, etc. as a proof-of-concept that elF2?AA cell failure is due to protein misfolding and that agents known to improve ER protein folding will improve function of cells pressured by proinsulin synthesis. Aim 2: Proinsulin misfolding in the ER causes Ca2+ leak to mitochondria, leading to oxidative stress. a. Does p58IPKdeficiency cause proinsulin misfolding in the ER to disrupt mitochondrial function and generate oxidative stress? Proinsulin synthesis, folding and trafficking, Ca^* imaging, mitochondrial membrane potential and ROS production in islets as well as in murine immortalized cell lines from p58IPK+/+ and p58IPK+/+ mice +/- glucose stimulation will be analyzed. b. Can SERCA overexpression improve insulin secretion and cell function in p58IPK+/+ cells and islets? c. Can cyclophilin D knockdown or deletion (Ppif-/-) prevent cell failure in p58IPK+/+ cells or mice, respectively? d. Can interventions in Id above improve function of p58IPK+/+ islets? For 2b-d, analyses will include methods described in 2a. Aim 3: IREI? and ATF6? provide overlapping functions to promote SRP-dependent ribosome and mRNA recruitment to the ER membrane during glucose stimulation and increase ER protein-folding capacity. a. How does Ire1? change membrane association of mRNAs? b. Can antioxidants, cyclosporine A, or chemical chaperones improve ire1?-/- cell function and change mRNA cellular localization? c. Is Atfd? and/or Atf6 deletion detrimental to cells upon Ire1?-/- deletion, HFD feeding, or Akita mutation?